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Summary Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX+/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo. Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye., Highlights • Longitudinal imaging of single fluorescently tagged donor photoreceptors in primates • Donor photoreceptor migration and neurite outgrowth were detected in the living eye • In a photoreceptor ablation model, donor photoreceptors migrated to the OPL • Donor photoreceptors structurally made synaptic connections with host bipolar cells, In this article, David Williams and colleagues demonstrate the capability of fluorescence adaptive optics imaging that allows longitudinal tracking of the behavior of transplanted photoreceptors at a single-cell level in living primate eyes. This study shows transplanted photoreceptors migrated to the outer plexiform layer through the laser lesion sites and often extended neurites containing presynaptic proteins toward the host inner nuclear layer.

At the cellular-scale, there was a strong correlation between the mean fluorescence lifetime using excitation wavelengths 532 and 473 nm with adaptive optics fluorescence lifetime ophthalmoscopy (FLIO), simplifying the comparison of AOFLIO and clinical FLIO.

This erratum corrects an error in Table 4 of our paper [Biomed. Opt. Express 13, 1737 (2022)10.1364/BOE.451628].

Fluorescence lifetime imaging has demonstrated promise as a quantitative measure of cell health. Adaptive optics two-photon excited fluorescence (TPEF) ophthalmoscopy enables excitation of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle, providing in vivo visualization of retinal structure and function at the cellular scale. Combining these technologies revealed that macaque cones had a significantly longer mean TPEF lifetime than rods at 730 nm excitation. At 900 nm excitation, macaque photoreceptors had a significantly longer mean TPEF lifetime than the retinal pigment epithelium layer. AOFLIO can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes.

Purpose The development of new approaches to human vision restoration could be greatly accelerated with the use of nonhuman primate models; however, there is a paucity of primate models of outer retina degeneration with good spatial localization. To limit ablation to the photoreceptors, we developed a new approach that uses a near-infrared ultrafast laser, focused using adaptive optics, to concentrate light in a small focal volume within the retina. Methods In the eyes of eight anesthetized macaques, 187 locations were exposed to laser powers from 50 to 210 mW. Laser exposure locations were monitored for up to 18 months using fluorescein angiography (FA), optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), adaptive optics scanning laser ophthalmoscope (AOSLO) reflectance imaging, two-photon excited fluorescence (TPEF) ophthalmoscopy, histology, and calcium responses of retinal ganglion cells. Results This method produced localized photoreceptor loss with minimal axial spread of damage to other retinal layers, verified by in-vivo structural imaging and histologic examination, although in some cases evidence of altered autofluorescence was found in the adjacent retinal pigment epithelium (RPE). Functional assessment using blood flow imaging of the retinal plexus and calcium imaging of the response of ganglion cells above the photoreceptor loss shows that inner retinal circuitry was preserved. Conclusions Although different from a genetic model of retinal degeneration, this model of localized photoreceptor loss may provide a useful testbed for vision restoration studies in nonhuman primates. Translational relevance With this model, a variety of vision restoration methods can be tested in the non-human primate.

The intrinsic fluorescence properties of lipofuscin – naturally occurring granules that accumulate in the retinal pigment epithelium – are a potential biomarker for the health of the eye. A new modality is described here which combines adaptive optics technology with fluorescence lifetime detection, allowing for the investigation of functional and compositional differences within the eye and between subjects. This new adaptive optics fluorescence lifetime imaging ophthalmoscope was demonstrated in 6 subjects. Repeated measurements between visits had a minimum intraclass correlation coefficient of 0.59 Although the light levels were well below maximum permissible exposures, the safety of the imaging paradigm was tested using clinical measures; no concerns were raised. This new technology allows for in vivo adaptive optics fluorescence lifetime imaging of the human RPE mosaic.

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Retinal function has long been studied with psychophysical methods in humans, whereas detailed functional studies of vision have been conducted mostly in animals owing to the invasive nature of physiological approaches. There are exceptions to this generalization, for example, the electroretinogram. This review examines exciting recent advances using in vivo retinal imaging to understand the function of retinal neurons. In some cases, the methods have existed for years and are still being optimized. In others, new methods such as optophysiology are revealing novel patterns of retinal function in animal models that have the potential to change our understanding of the functional capacity of the retina. Together, the advances in retinal imaging mark an important milestone that shifts attention away from anatomy alone and begins to probe the function of healthy and diseased eyes.

Purpose Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. Methods Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. Results All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Muller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. Conclusions This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes.

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinically-relevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.

Nonlinear microscopy has found its niche for anatomical and physiological imaging of living tissue. This chapter treats the technique’s fusion with in vivo high-resolution retinal imaging, which enables noninvasive longitudinal studies. Successful implementation requires consideration of the eye’s optical properties as well as light safety aspects. We report the latest achievements in this research field and highlight the future potential of nonlinear retinal imaging.

As one of the most relied upon senses, an understanding of the visual system is essential. There are two focussing lenses, the cornea and the crystalline lens, contributing approximately 40D and 20D, respectively, to the total power of the eye. The anterior surface of the cornea is shaped like a prolate ellipsoid (Kiely et al, 1982).

Computational modeling of laser-tissue interaction in the retina can be used to predict the thermal response to a laser exposure. For in vivo photochemical damage threshold experiments, where temperature rise in retinal tissue cannot be conveniently measured, modeling was used to predict retinal temperature response. Laser exposures from sixteen in vivo experiments with wavelengths in the visible band were selected to represent a wide range of powers and exposure durations. It is hypothesized that photochemical damage mechanisms were responsible for changes to subjects’ retinal tissue and that thermal response is not a significant contribution to this observed damage.Results from the model predicted peak temperature responses of less than one Kelvin in the RPE layer of the retina where the largest temperature rise would be expected. These findings support the claim that changes to retinal tissue were not directly caused by a thermally induced mechanism.Computational modeling of laser-tissue interaction in the retina can be used to predict the thermal response to a laser exposure. For in vivo photochemical damage threshold experiments, where temperature rise in retinal tissue cannot be conveniently measured, modeling was used to predict retinal temperature response. Laser exposures from sixteen in vivo experiments with wavelengths in the visible band were selected to represent a wide range of powers and exposure durations. It is hypothesized that photochemical damage mechanisms were responsible for changes to subjects’ retinal tissue and that thermal response is not a significant contribution to this observed damage.Results from the model predicted peak temperature responses of less than one Kelvin in the RPE layer of the retina where the largest temperature rise would be expected. These findings support the claim that changes to retinal tissue were not directly caused by a thermally induced mechanism.

Insertion of light-gated channels into inner retina neurons restores neural light responses, light evoked potentials, visual optomotor responses and visually-guided maze behavior in mice blinded by retinal degeneration. This method of vision restoration bypasses damaged outer retina, providing stimulation directly to retinal ganglion cells in inner retina. The approach is similar to that of electronic visual protheses, but may offer some advantages, such as avoidance of complex surgery and direct targeting of many thousands of neurons. However, the promise of this technique for restoring human vision remains uncertain because rodent animal models, in which it has been largely developed, are not ideal for evaluating visual perception. On the other hand, psychophysical vision studies in macaque can be used to evaluate different approaches to vision restoration in humans. Furthermore, it has not been possible to test vision restoration in macaques, the optimal model for human-like vision, because there has been no macaque model of outer retina degeneration. In this study, we describe development of a macaque model of photoreceptor degeneration that can in future studies be used to test restoration of perception by visual prostheses. Our results show that perceptual deficits caused by focal light damage are restricted to locations at which photoreceptors are damaged, that optical coherence tomography (OCT) can be used to track such lesions, and that adaptive optics retinal imaging, which we recently used for in vivo recording of ganglion cell function, can be used in future studies to examine these lesions.

Susana Marcos et al. -- 31 pags., 13 figs., Adaptive optics is a relatively new field, yet it is spreading rapidly and allows new questions to be asked about how the visual system is organized. The editors of this feature issue have posed a series of question to scientists involved in using adaptive optics in vision science. The questions are focused on three main areas. In the first we investigate the use of adaptive optics for psychophysical measurements of visual system function and for improving the optics of the eye. In the second, we look at the applications and impact of adaptive optics on retinal imaging and its promise for basic and applied research. In the third, we explore how adaptive optics is being used to improve our understanding of the neurophysiology of the visual system., John S. Werner: NIH Grants R01 EY 024239, AG04058, P-30 EY012576 and Research to Prevent Blindness. Susana Marcos: European Research Council under the European Union’s Seventh Framework Program (FP/2007–2013)/ERC Grant Agreement [ERC- 2011-AdC 294099]. Spanish Government Grants FIS2011-25637 & FIS2014-56643-R, Spanish Government FPU and FPI Predoctoral Fellowships, CSIC JAE-Pre program., Pablo Artal: European Research Council Advanced Grant ERC-2013-AdG-339228 (SEECAT) and the Spanish SEIDI, grant FIS2013-41237-R. David Atchison: Australian Research Council Discovery Grants DP110102018 and DP140101480. Karen Hampson: UK College of Optometrists; EPSRC (Engineering and Physical Sciences Research Council), Grants EP/ D036550/1and EP/G015473/1. Linda Lundstrom: Swedish Agency for Innovation Systems (VINNMER 2008–00992) and Swedish Research Council (621-2011-4094). Geungyoung Yoon: NIH/NEI, NYSTAR. Stephen Burns and Ann Elsner NIH Grants EY007624, EY026105, EY024315 and EY004395. P30EY019008 and Foundation Fighting Blindness., Joseph Carroll: NIH R01EY017607, Foundation Fighting Blindness, and Research to Prevent Blindness. Stacey S. Choi and Nathan Doble: support by NIH EY020901 and Department of Defense (DoD) Telemedicine and Advanced Technology Research Center (TATRC) grant W81XWH-10-1-0738. Alfredo Dubra: the Burroughs Wellcome Fund, Research to Prevent Blindness, Glaucoma Research Foundation and National Eye Institute. Ravi Jonnal NEI K99 EY 026068. Donald Miller NEI R01-EY018339 and P30-EY019008. Michel Paques: Institut National de la Santé et de la Recherche Médicale (Contrat d’Interface 2011) the Agence Nationale de la Recherche (ANR-09-TECS-009, ANR-12-TECS-0015- 03 and LabEx LifeSenses ANR-10-LABX-65). Hannah Smithson and Laura K. Young: the John Fell Fund (University of Oxford), Fight for Sight, and the Wellcome Trust Institutional Strategic Support Fund. Yuhua Zhang: NIH Grants EY021903, EY024378, P30 EY003039) and NSF IIA-1539034., Melanie Campbell: Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada Collaborative Health Research Program. Jennifer Hunter: NIH R44 AG043645, R01 EY022371, R01 EY004367, P30 EY001319, and BRP-EY014375. Also supported by an unrestricted grant to the University of Rochester Department of Ophthalmology from Research to Prevent Blindness, New York, New York. Andrew Metha: Supported by an Australia Research Council (ARC) Discovery Project Grant (DP0984649), an ARC Discovery Early Career Researcher Award (DE120101931), an A.E. Rowden White Foundation benevolent bequest, and the University of Melbourne Interdis- ciplinary Seed Fund. Grazyna Palczewska: NIH NEI R44 AG043645, European Private Foundation. Jesse Schallek: NIH Grants EY023496-01, EY014375, EY001319, The Schmitt Program on Integrative Brain Research, the Research to Prevent Blindness Stein Innovation award, Research to Prevent Blindness Career Development award (Schallek), an unrestricted grant to the University of Rochester Department of Ophthalmology from Research to Prevent Blindness, New York, New York, and Canon Inc. Lawrence Sincich: National Eye Institute, Eyesight Foundation of Alabama.William H. Merigan: NIH R44 AG043645, R01 EY021166, and P30 EY001319. Also by an unrestricted grant to the University of Rochester Department of Ophthalmology from Research to Prevent Blindness, New York, New York.

Observations of RPE disruption and autofluorescence (AF) photobleaching at light levels below the ANSI photochemical maximum permissible exposure (MPE) (Morgan et al., 2008) indicates a demand to modify future light safety standards to protect the retina from harm. To establish safe light exposures, we measured the visible light action spectrum for RPE disruption in an in vivo monkey model with fluorescence adaptive optics retinal imaging. Using this high resolution imaging modality can provide insight into the consequences of light on a cellular level and allow for longitudinal monitoring of retinal changes. The threshold retinal radiant exposures (RRE) for RPE disruption were determined for 4 wavelengths (460, 488, 544, and 594 nm). The anaesthetized macaque retina was exposed to a uniform 0.5° × 0.5° field of view (FOV). Imaging within a 2° × 2° FOV was performed before, immediately after and at 2 week intervals for 10 weeks. At each wavelength, multiple RREs were tested with 4 repetitions each to determine the threshold for RPE disruption. For qualitative analysis, RPE disruption is defined as any detectable change from the pre exposure condition in the cell mosaic in the exposed region relative to the corresponding mosaic in the immediately surrounding area. We have tested several metrics to evaluate the RPE images obtained before and after exposure. The measured action spectrum for photochemical RPE disruption has a shallower slope than the current ANSI photochemical MPE for the same conditions and suggests that longer wavelength light is more hazardous than other measurements would suggest.

It has been known since the 19th century that light is captured by visual pigments in the outer segments (OS) of photoreceptor cells.1,2 Experiments performed by Wald3 and Hubbard and Wald4 revealed that visual pigment molecules are composed of two components, namely an opsin protein and a chromophore, 11-cis-retinal.3,4 Upon absorption of a photon by the pigment, the chromophore isomerizes to all-trans-retinal, which subsequently is released from the opsin through a series of intermediate steps.5 This initiates the regeneration of photopigment through the visual (retinoid) cycle.6 During regeneration, all-trans-retinal is reduced to all-trans-retinol,7 which subsequently is transferred from OS to RPE cells8 and also to Muller cells, which contribute to photopigment regeneration in cones.9–12 Measurement of the photopigment regeneration rate has been used as a marker for disease in photoreceptors and the RPE.13 To monitor pigment regeneration directly in the living human eye, rhodopsin densitometry has been used.14–16 However, interpretation of densitometry data is complicated by the contribution of intrinsic optical signals to observed changes in reflectance, which currently are inexplicable within the current model of pigment regeneration kinetics.17–19 Retinoid absorption and fluorescence properties also can be exploited to track retinoid concentrations following light exposure. Ultraviolet (UV) light has been used to excite autofluorescence from all-trans-retinol to track the visual cycle in isolated cells from various animal models.20–23 However, different species metabolize retinoids at different rates, complicating direct comparisons of regeneration rates in animal versus human retina.13 The physiology and anatomy of nonhuman primate retina, particularly old-world monkeys,24 is similar to that in humans, making them an appropriate animal model. However, little is known about the kinetics of intermediate stages of the retinoid cycle in living primates. For in vivo imaging, optical techniques relying on single-photon fluorescence excitation of retinoids cannot be used in primates because UV light is outside the spectral barrier for transmission through the optics of the eye.25 Two-photon fluorescence (TPF) imaging26,27 overcomes these spectral barriers and offers the potential for a noninvasive approach for studying visual function at a cellular scale in the living eye. Previously, we have demonstrated TPF imaging of photoreceptors and RPE in the mouse eye28–31 and an increased fluorescence over time in photoreceptors of the macaque.32 From the point of view of translating this technique to human imaging, its use depends on identifying and characterizing the dominant source(s) of time-variable autofluorescence in primate eyes, which could emanate from one or more among a list of candidate fluorophores. Examples include retinoids, NAD(P)H, FAD, collagen, elastin, lipofuscin, and perhaps others.33–35 Previous reports on autofluorescence from the retina20,21,29,36 have identified all-trans-retinol as the dominant source of time-variable fluorescence in mammalian photoreceptors. To investigate this hypothesis in living primates, we used a new generation two-photon ophthalmoscope to characterize the TPF responses from individual photoreceptors under different conditions of light and dark adaptation.

Purpose Infrared ultrashort pulse lasers are becoming increasingly popular for applications in the living eye. However, safety standards are not yet well established. Here we investigate retinal damage close to threshold for this pulse regime in the living macaque eye. Methods Retinal radiant exposures between 214 and 856 J/cm2 were delivered to the photoreceptor layer with an ultrashort pulse laser (730 nm, 55 fs, 80 MHz) through a two-photon adaptive optics scanning light ophthalmoscope. Retinal exposures were followed up immediately after and over several weeks with high-resolution reflectance and two-photon excited fluorescence ophthalmoscopy, providing structural and functional information. Results Retinal radiant exposures of 856 J/cm2 resulted in permanent S cone damage. Immediately after the exposure, the affected cones emitted about 2.6 times less two-photon excited fluorescence (TPEF) and showed an altered TPEF time course. Several weeks after the initial exposure, S cone outer and inner segments had disappeared. The space was filled by rods in the peripheral retina and cones near the fovea. Conclusion Interestingly, S cones are the receptor class with the lowest sensitivity in the near-infrared but are known to be particularly susceptible to ultraviolet and blue light. This effect of selective S cone damage after intense infrared ultrashort pulse laser exposure may be due to nonlinear absorption and distinct from pure thermal and mechanical mechanisms often associated with ultrashort pulse lasers.

The degree of polarization (DOP) of the light reflected from the optic nerve head has been assessed by means of a polarimetric scanning laser ophthalmoscope as a function of the age of the participants. Four fundus images corresponding to independent polarization states in the recording pathway were used to compute the spatially-resolved DOP. This was not uniform across the optic nerve head and depended on both the location and the participant's age. Along a peripapillary annulus the DOP followed a double-peak pattern. Moreover, the values along this annulus decreased significantly with increasing age. This depolarization appears to originate in part in the retinal nerve fiber layer. Detailed age-dependent knowledge of the ocular depolarization properties may help to improve clinical diagnosis of the retinal nerve fiber layer.

Chicks develop refractive errors in response to defocusing lenses. Mean ocular refractions (MOR) and astigmatism were measured in chicks that developed myopia in response to unilaterally applied negative lenses (-30 D, n = 19; -15 D, n = 16). The MOR of the control eyes emmetropized with age and both goggle powers were fully compensated given enough time. The initial slopes of plots of MOR with age are not significantly different between inducing lens powers. Astigmatism in control eyes decreased significantly with age. In the presence of -30 D lenses, astigmatism significantly increased with age due to an exponential increase in JCC45 (oblique Jackson crossed cylinder). Although not statistically significant, a similar but smaller trend was observed for the -15 D lenses. Thus, oblique astigmatism results during myopia induction by high-powered lenses. This induced astigmatism is correlated with MOR. These results are consistent with emmetropization of astigmatism, which is disrupted by high-powered spher...

Stargardt disease (STGD1) is characterized by macular atrophy and flecks in the retinal pigment epithelium. The causative ABCA4 gene encodes a protein localizing to photoreceptor outer segments. The pathologic steps by which ABCA4 mutations lead to clinically detectable retinal pigment epithelium changes remain unclear. We investigated early STGD1 using adaptive optics scanning light ophthalmoscopy.Adaptive optics scanning light ophthalmoscopy imaging of 2 brothers with early STGD1 and their unaffected parents was compared with conventional imaging. Cone and rod spacing were increased in both patients (P .001) with a dark cone appearance. No foveal cones were detected in the older brother. In the younger brother, foveal cones were enlarged with low density (peak cone density, 48.3 × 103 cones/mm2). The ratio of cone to rod spacing was increased in both patients, with greater divergence from normal approaching the foveal center, indicating that cone loss predominates centrally and rod loss increases peripherally. Both parents had normal photoreceptor mosaics. Genetic testing revealed 3 disease-causing mutations.This study provides in vivo images of rods and cones in STGD1. Although the primary clinical features of STGD1 are retinal pigment epithelial lesions, adaptive optics scanning light ophthalmoscopy reveals increased cone and rod spacing in areas that appear normal in conventional images, suggesting that photoreceptor loss precedes clinically detectable retinal pigment epithelial disease in STGD1.

The retina contains endogenous fluorophores (e.g. NADH, retinoids and others) that are natural markers for physiology and morphology. Adaptive optics assisted two-photon excitation was employed to record fluorescence from these molecules throughout the retina in living macaques.

Understanding the control of eye growth may lead to the prevention of nearsightedness (myopia). Chicks develop refractive errors in response to defocusing lenses by changing the rate of eye elongation. Changes in optical image quality and the optical signal in lens compensation are not understood. Monochromatic ocular aberrations were measured in 16 chicks that unilaterally developed myopia in response to unilateral goggles with -15D lenses and in 6 chicks developing naturally. There is no significant difference in higher-order root mean square aberrations (RMSA) between control eyes of the goggled birds and eyes of naturally developing chicks. Higher-order RMSA for a constant pupil size exponentially decreases in the chick eye with age more slowly than defocus. In the presence of a defocusing lens, the exponential decrease begins after day 2. In goggled eyes, asymmetric aberrations initially increase significantly, followed by an exponential decrease. Higher-order RMSA is significantly higher in goggled eyes than in controls. Equivalent blur, a new measure of image quality that accounts for increasing pupil size with age, exponentially decreases with age. In goggled eyes, this decrease also occurs after day 2. The fine optical structure, reflected in higher-order aberrations, may be important in understanding normal development and the development of myopia.

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

Purpose Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol. Methods Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye's pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics. Results Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds. Conclusions This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.

PURPOSE Advances in retinal imaging have led to the discovery of long-lasting retinal changes caused by light exposures below published safety limits, including disruption of the RPE. To investigate the functional consequences of RPE disruption, we combined adaptive optics ophthalmoscopy with retinal densitometry. METHODS A modified adaptive optics scanning light ophthalmoscope (AOSLO) measured the apparent density and regeneration rate of rhodopsin in two macaques before and after four different 568-nm retinal radiant exposures (RREs; 400-3200 J/cm(2)). Optical coherence tomography (OCT) was used to measure the optical path length through the photoreceptor outer segments before and after RPE disruption. RESULTS All tested RREs caused visible RPE disruption. Apparent rhodopsin density was significantly reduced following 1600 (P = 0.01) and 3200 J/cm(2) (P = 0.007) exposures. No significant change in apparent density was observed in response to 800 J/cm(2). Surprisingly, exposure to 400 J/cm(2) showed a significant increase in apparent density (P = 0.047). Rhodopsin recovery rate was not significantly affected by these RREs. Optical coherence tomography measurements showed a significant decrease in the optical path length through the photoreceptor outer segments for RREs above 800 J/cm(2) (P < 0.001). CONCLUSIONS At higher RREs, optical path length through the outer segments was reduced. However, the rate of photopigment regeneration was unchanged. While some ambiguity remains as to the correlation between measured reflectivity and absolute rhodopsin density; at the lowest RREs, RPE disruption appears not to be accompanied by a loss of apparent rhodopsin density, which would have been indicative of functional loss.

Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.

Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.

Two-photon ophthalmoscopy has potential for in vivo assessment of function of normal and diseased retina. However, light safety of the sub-100 fs laser typically used is a major concern and safety standards are not well established. To test the feasibility of safe in vivo two-photon excitation fluorescence (TPEF) imaging of photoreceptors in humans, we examined the effects of ultrashort pulsed light and the required light levels with a variety of clinical and high resolution imaging methods in macaques. The only measure that revealed a significant effect due to exposure to pulsed light within existing safety standards was infrared autofluorescence (IRAF) intensity. No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptors and retinal pigment epithelium appeared normal in adaptive optics images. No effect of repeated exposures on TPEF time course was detected, suggesting that visual cycle function was maintained. If IRAF reduction is hazardous, it is the only hurdle to applying two-photon retinal imaging in humans. To date, no harmful effects of IRAF reduction have been detected.

Purpose To demonstrate the feasibility of in vivo measurements of cones and their distributions as a function of normal growth without adaptive optics (AO) and also discuss the potential advantages and disadvantages of AO imaging in the chick, an animal model of myopia. Methods Chicks were obtained on the day of hatching. Axial length and retinoscopy measurements were performed on days 0 and 14. Chicks were imaged on the day of hatching and 14 days later in a custom-built confocal scanning laser ophthalmoscope. Angular densities, linear cone spacings, and cone packing arrangements were determined. Results Four subarrays of hexagonally packed cones were identified on both days and, from their angular spacings, appear to correspond to different cone types. There were no significant changes in angular cone density with growth and linear spacings of cones increased with growth. This is true for both overall densities and those of the cone subtypes. There was no change in the percent of hexagonally packed cones with growth. Conclusions Cones can be imaged longitudinally in vivo in the awake chick. The packing arrangement of cones is 40% hexagonally packed. Although AO is not necessary to visualize the cones, including the subarrays of like cones, some closely spaced cones of different types may not be resolved. Most importantly, there is a need to use a larger pupil with growth to maintain the same linear resolution in the larger eye. Novel longitudinal imaging techniques and methods in animal models are shown here to give insights into normal development and, in future, will give insights into visual disorders and diseases, including myopia.

Longitudinal chromatic aberration (LCA) in mouse eyes is detrimental for signal efficiency in in vivo two-photon imaging. Furthermore, LCA varies from eye to eye. Here, we propose and verify a solution for tunable achromatization.

The photoreceptor/RPE complex must maintain a delicate balance between maximizing the absorption of photons for vision and retinal image quality while simultaneously minimizing the risk of photodamage when exposed to bright light. We review the recent discovery of two new effects of light exposure on the photoreceptor/RPE complex in the context of current thinking about the causes of retinal phototoxicity. These effects are autofluorescence photobleaching in which exposure to bright light reduces lipofuscin autofluorescence and, at higher light levels, RPE disruption in which the pattern of autofluorescence is permanently altered following light exposure. Both effects occur following exposure to visible light at irradiances that were previously thought to be safe. Photopigment, retinoids involved in the visual cycle, and bisretinoids in lipofuscin have been implicated as possible photosensitizers for photochemical damage. The mechanism of RPE disruption may follow either of these paths. On the other hand, autofluorescence photobleaching is likely an indicator of photooxidation of lipofuscin. The permanent changes inherent in RPE disruption might require modification of the light safety standards. AF photobleaching recovers after several hours although the mechanisms by which this occurs are not yet clear. Understanding the mechanisms of phototoxicity is all the more important given the potential for increased susceptibility in the presence of ocular diseases that affect either the visual cycle and/or lipofuscin accumulation. In addition, knowledge of photochemical mechanisms can improve our understanding of some disease processes that may be influenced by light exposure, such as some forms of Leber's congenital amaurosis, and aid in the development of new therapies. Such treatment prior to intentional light exposures, as in ophthalmic examinations or surgeries, could provide an effective preventative strategy.

Adaptive optics imaging of cone photoreceptors has provided unique insight into the structure and function of the human visual system and has become an important tool for both basic scientists and clinicians. Recent advances in adaptive optics retinal imaging instrumentation and methodology have allowed us to expand beyond cone imaging. Multi-wavelength and fluorescence imaging methods with adaptive optics have allowed multiple retinal cell types to be imaged simultaneously. These new methods have recently revealed rod photoreceptors, retinal pigment epithelium (RPE) cells, and the smallest retinal blood vessels. Fluorescence imaging coupled with adaptive optics has been used to examine ganglion cells in living primates. Two-photon imaging combined with adaptive optics can evaluate photoreceptor function non-invasively in the living primate retina.

Advances in adaptive optics scanning laser ophthalmoscopy have made it possible to image the living retina with better resolution than ever before, making it possible to see the smallest retinal cells including cones at the foveal center and individual rods. In addition, we have demonstrated the feasibility of safe in vivo two-photon fluorescence imaging, which allows us to explore molecules in the retina that were previously inaccessible in the living eye.

Estrogen receptor (ER)-negative breast cancer shows a higher incidence in women of African ancestry compared to women of European ancestry. In search of common risk alleles for ER-negative breast cancer, we combined genome-wide association study (GWAS) data from women of African ancestry (1,004 ER-negative cases and 2,745 controls) and European ancestry (1,718 ER-negative cases and 3,670 controls), with replication testing conducted in an additional 2,292 ER-negative cases and 16,901 controls of European ancestry. We identified a common risk variant for ER-negative breast cancer at the TERT-CLPTM1L locus on chromosome 5p15 (rs10069690: per-allele odds ratio (OR) = 1.18 per allele, P = 1.0 x 10(-10)). The variant was also significantly associated with triple-negative (ER-negative, progesterone receptor (PR)-negative and human epidermal growth factor-2 (HER2)-negative) breast cancer (OR = 1.25, P = 1.1 x 10(-9)), particularly in younger women (

Background Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed. Methods We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue. Results Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections. Conclusion This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.

PURPOSE. The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. METHODS. Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. RESULTS. Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics. Adaptive optics correction reduced the total root mean square wavefront error, on average, from 0.30 m to 0.05 m (measured at 904 nm, 1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was approximately 1.84 m, approximately 82% greater than the FWHM of the diffraction-limited LSF. CONCLUSIONS. With perfect aberration compensation, the in vivo resolution in the rat eye could be approximately 2 greater than that in the human eye because of its large numerical aperture (0.43). Although the fAOSLO corrects a substantial fraction of the rat eye’s aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using adaptive optics to investigate the rodent eye in vivo with high resolution. (Invest Ophthalmol Vis Sci. 2009;50:5872‐5879) DOI:10.1167/iovs.09-3675

In the simplest model of eye growth, the ocular optics uniformly scale upwards, as do monochromatic higher-order aberrations (HOA) and linear blur on the retina. However, measured HOA remain constant or decrease with growth in some species. A new model, which holds HOA and the associated linear blur on the retina constant, was used to predict changes in HOA and resulting image quality on the retina during growth, in each of chick, monkey, and human. Models used rates of growth in each of the three species. Angular optical quality on the retina due to HOA, and its metrics improved, in contrast to the constancy predicted by uniform scaling. The model with constant linear HOA blur predicts well the improvement in human optical quality between infant and adult. Overall, in chick and monkey, angular blur improves at a rate faster than that predicted by the constant linear blur model, implying that linear retinal blur due to HOA decreases with age. On the other hand, in chick, angular blur due to third-order aberrations decreased at a rate predicted by the constant linear blur model. Growth changes in retinal blur due to HOA are species dependent but can be better understood by comparison with the new model predictions.

Using a fluorescence-equipped adaptive optics scanning laser ophthalmoscope, we have discovered retinal changes in the in vivo macaque retina resulting from exposure to visible light at levels below the ANSI maximum permissible exposure.

The American National Standards Institute (ANSI) provides guidelines for the safe use of lasers from which maximum permissible exposures of laser light are calculated. This standard is used to limit ocular light exposures and to assess the safety of retinal imaging experiments. Using in vivo adaptive optics scanning laser ophthalmoscopy equipped with fluorescence imaging capabilities, we have observed retinal changes in the macaque retina caused by visible light exposures that are below the ANSI safety standard. Specifically, we observed an immediate decrease in the autofluorescence intensity of the retinal pigment epithelial cells at the exposure location, followed by long-term full recovery or permanent retinal damage. The AF reduction was observed with exposure to 488 nm or 568 nm light. We have demonstrated the photochemical nature of the AF reduction. Experimental evidence suggests that multiple lipofuscin fluorophores are involved in the reduction of AF. Since the mechanism causing the AF reduction is currently unknown, it remains unclear whether these effects are detrimental to retinal health. Even if the AF reduction is not hazardous, our results encourage a re-evaluation of the standards for avoiding photochemical damage from exposures in the middle of the visible spectrum.The American National Standards Institute (ANSI) provides guidelines for the safe use of lasers from which maximum permissible exposures of laser light are calculated. This standard is used to limit ocular light exposures and to assess the safety of retinal imaging experiments. Using in vivo adaptive optics scanning laser ophthalmoscopy equipped with fluorescence imaging capabilities, we have observed retinal changes in the macaque retina caused by visible light exposures that are below the ANSI safety standard. Specifically, we observed an immediate decrease in the autofluorescence intensity of the retinal pigment epithelial cells at the exposure location, followed by long-term full recovery or permanent retinal damage. The AF reduction was observed with exposure to 488 nm or 568 nm light. We have demonstrated the photochemical nature of the AF reduction. Experimental evidence suggests that multiple lipofuscin fluorophores are involved in the reduction of AF. Since the mechanism causing the AF reduction ...